Megakaryocyte (MK) maturation and platelet production has been long thought to be regulated by lineage specific humoral growth factors in a manner similar to cytokines that induce erythrocyte (erythropoietin) and granulocyte (G-CSF) expansion and maturation. Platelets are responsible for the prevention of bleeding in response to vascular injury. Therefore, platelet production is a vital component of hematopoietic regulation. Patients undergoing chemotherapy or bone marrow transplantation usually experience severely depressed platelet levels (thrombocytopenia) which may result in life threatening bleeding episodes. Several known growth factors and cytokines have been found to stimulate megakaryocytes and platelet production but most are pleiotropic both in vitro and in vivo (IL-3, IL-6, IL-11, SCF). Plasma, serum and urine from thrombocytopenic dogs and humans have been found to contain growth factors that have specific megakaryopoietic and thrombopoietic activities distinct from all known cytokines. These factors have been termed Meg-CSF, MK-CSF, megakaryocyte growth and development factor (MGDF), megakaryopoietin, and thrombopoietin but the molecular structure has not been identified until recently.
The identification of the thrombopoietic cytokine, c-mpl ligand, originated with the identification of a myeloproliferative leukemia virus (MPLV, Wending et al., Virology 149:242-246 [1986]). Mice infected with this virus gave rise to multi-lineage myeloproliferation. Subsequent studies (Souyri et al., Cell 63:1137-1147, [1990]) demonstrated that the retrovirus encoded an oncogene (v-mpl) that when fused with viral envelope gene gave rise to a membrane anchored protein that resembles the cytoplasmic domain of the hematopoietic growth factor receptor family. V-mpl was used to probe both human and murine RNA libraries for homologous genes. Clones were identified in both species and termed c-mpl (Vigon et al., PNAS 89:5640-5644 [1992], Vigon et al., Oncogene 8:2607-2615 [1993]). C-mpl is a member of the cytokine receptor super-family with regions of homology to mIL-5rc, IL3rc, IL4rc, mEPOrc and mGCSFrc. A chimera of the intracellular domain of c-mpl and the extracellular domain of hIL-4rc was transfected into a growth factor dependent cell line (BaF3). Once transfected, the cells proliferated in response to hIL4 indicating that the c-mpl cytoplasmic domain was fully sufficient to transduce a proliferative signal (Skoda et al., EMBO J. 12(7):2645-2653 [1993]).
Message for c-mpl was found in a number of hematopoietic cell lines using reverse transcriptase polymerase chain reaction (RT-PCR) including the pluripotential cell lines TF-1, Mo-7E, UT-7 and KU812; and erythro/megakaryocytic cell lines HEL, DAMI and K153. Transcripts were also identified in bone marrow, fetal liver, megakaryocytes, platelets and CD34+enriched cells (Methia et al., Blood 82(5):1395-1401 [1993]).
The identification of a putative receptor triggered several investigative teams to search for a naturally occurring ligand for c-mpl. In June of 1994 several simultaneous publications reported on a ligand that bound to c-mpl and had megakaryocytopoietic properties (de Sauvage et al., Nature 369:533-539 [1994]; Lok et al., Nature 369:565-568 [1994]; Wendling et al., Nature 369:571-574 [1994] and Bartley et al., Cell 77:1117-1124 [1994]). The ligand referred to as c-mpl ligand, Megakaryocyte Growth and Development Factor (MGDF) or thrombopoietin (TPO) is a peptide with a predicted molecular mass of 35,000 kDa. The protein has a two domain structure with an amino-terminal domain (153 amino acids) with homology to erythropoietin and a carboxy-terminus rich in serine, threonine and proline residues which also contains several glycosylation sites. There are two potential arginine cleavage sites resulting in two shorter peptides of 25 kDa and 31 kDA forms both of which are biologically active. There is high inter-species homology between human, murine, porcine, canine, rat and rabbit c-mpl ligand and most forms are active on all species tested.
C-mpl ligand has been shown to stimulate the differentiation of CD34+ cells into cells with megakaryocyte characteristics. CD34+ cells, in the presence of c-mpl ligand, underwent endomitosis (Kaushansky et al., Nature 369:568-571 [1994]), expressed the megakaryocyte lineage specific cell surface antigen CD41a and had morphology characteristic of megakaryocytes. In vivo administration of c-mpl ligand gave rise to increased circulating platelets in normal mice (Lok et al., Nature 369:565-568 [1994]). C-mpl deficient mice generated by gene targeting demonstrated a 85% decrease in circulating platelets and megakaryocytes but had normal amounts of other hematopoietic lineages (Gurney et al., Science 265:1445-1447 [1994]). Absolute thrombocytopenia was not observed in these animals indicating that other cytokines may have some activity in expansion of the MK lineage.
Studies to date show that c-mpl ligand is a cytokine with specific activity on the maturation of megakaryocytes and in platelet production. Other cytokines have been shown to have activity on megakaryocyte expansion and differentiation, including IL-3, IL-6, IL-11 and c-kit ligand. Recent studies have demonstrated that these cytokines (with the exception of IL-3) act by stimulating the production of c-mpl ligand and do not have megakaryocyte stimulating activity by themselves (Kaushansky et al., PNAS 92:3234-3236 [1995]).
GB 2,285,446 relates to the c-mpl ligand (thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
The ability of c-mpl ligand to stimulate the proliferation and maturation of megakaryocytes and production of platelets indicates that c-mpl ligand may have therapeutic use in restoring circulating platelets to normal amounts in those cases where the number of platelets have been reduced due to diseases or therapeutic treatments such as radiation and/or chemotherapy.
EP 675,201 A1 relates to the c-mpl ligand (Megakaryocyte growth and development factor [MGDF]), allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof. The proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
A previously published abstract (Eaton et al., Blood 84(10) Suppl. abstract 948, [1994]) reported c-DNA for an alternative splice form of c-mpl ligand identified in man, dog and mouse. The encoded protein has 4 amino deletion at position aa112-115. Although this molecule showed no activity in their bioassays, mRNA for this variant was found to be abundant in all three species indicating that it may be a naturally occurring alternative form of c-mpl ligand. Contrary to the previously published report, we found that the 1-153 .DELTA.112-115 c-mpl ligand and the 1-332 .DELTA.112-115 c-mpl ligand were biological active.